It has been shown that peptides containing non-proteinogenic backbone structures have novel conformational and biomolecular recognition properties as well as altered in vivo and in vitro stability compared to a-peptides. Additionally, non-natural amino acid incorporation work using in vitro translation systems and chemically-charged tRNA have illustrated the enormous utility of altering backbone structure in studying the contributions of backbone conformational rigidity and hydrogen bonding to protein stability, catalysis, and regulation. Clearly, many important biomedical insights as well as medically relevant technologies can results from alteration of peptide backbone structure. Recent development of techniques for in vivo site-specific incorporation of non-natural amino acid moieties into proteins using engineered orthogonal tRNA/tRNA synthetase pairs has made possible a number of elegant protein structure/function studies and breakthrough bio-technological applications. However, all of the work done using orthogonal pairs has focused on changing the structures of amino acid side chains. This proposal focuses on the extension of the orthogonal tRNA/tRNA synthetase pair evolution methodology to develop in vivo systems in both E. coli and S. cerevisiae capable of site-specifically incorporating amino acids bearing N-?-methyl, C-?- methyl, and 3-structural changes, as well as a-hydroxy acids, into proteins and peptides. Previously used M. jannaschii amber suppressor tRNATyr/tyrosyl-tRNA synthetase orthogonal pair for use in E. coli, and E. coli tRNATyr/TyrRS and E. coli tRNALeu/leucyl-tRNA synthetase pairs for use in S. cerevisiae will be employed as starting points for engineering. Residues of tRNA synthetases in proximity to the ?-amino group and ?- and (3-pro-S protons of the bound amino acid, as deduced from relevant crystal structures, will be randomized. The resulting libraries will be subjected to several rounds of positive and negative selection in order to obtain evolved synthetases specific for each non-natural compound. Demonstration of efficient in vitro tRNA charging and in vivo incorporation of non-natural amino acids into model proteins by evolved synthetases will be demonstrated. Selected amino acids/analogues will be site-specifically incorporated into biomedically important target enzymes and the properties of each modified target will be compared to those of the wild-type to demonstrate the novel properties imparted by the modification. Relevance: Many disease states and therapies involve participation of proteins and/or peptides. The ability to understand and control protein/peptide structure and function is critical to developing new treatments for disease. This work will contribute a new tool for understanding and controlling protein structure and function.